The lung is a major dose-limiting tissue in radiation therapy. We have demonstrated in mice that intratracheal infection of plasmid liposomes or second generation replication-defective adenovirus constructs carrying the human manganese superoxide dismutase (MnSOD) transgene results in a significant decrease in both acute and chronic (alveolitis/fibrosis) damage by whole long irradiation. Both delivery systems demonstrated increased MnSOD mRNA levels in the airway prior to irradiation and decreased levels of transcripts for inflammatory cytokines that irradiation. We will now confirm the observations, and optimize in rigorous preclinical studies the efficiency and safety of pulmonary MnSOD transgene therapy using plasmid/liposomes for lung irradiation protection. Three specific are designed for proof of the principal that MnSOD transgene therapy will protect normal lung from irradiation damage. In the first specific aim C57BL/6J mice, which are heterozygous deletion recombinant-negative for murine MNSOD, will be compared will be compared with normal littermates for irradiation-induced organizing alveolitis. Our preliminary data show increased susceptibility of MnSOD heterozygous knockout mice to irradiation-induced alveolitis and are corroborated by the increased radiosensitivity in vitro of (-/-) and (-/+) human MnSOD transgene. In the second specific aim we will determine the level of over- expression of MnSOD which correlates with the relative lung radioresistance in FeVB/NHsd transgenic mice over-expressing the human MnSOD transgene in the airway linked to the lung parenchymal cell-specific SP1 (surfactant) promoter. These mice will be treated with additional intratracheal plasmid/liposome transgene therapy, and irradiation-induced alveolitis will be quantitated. In the third specific aim we will optimize the timing of intratracheal delivery of MnSOD plasmid/liposomes to protect the lungs of bilateral or unilateral lung irradiated C57/BL/6J, control, and orthotopic Lewis lung tumor-bearing mice in clinically relevant fractionated radiotherapy. Preliminary data demonstrate significant irradiation protection by MnSOD plasmid/liposome delivery of the human MnSOD transgene, the transcripts of which are detected in the airway but not in lung tumors by nested RT-PCR. These studies should optimize MnSOD- mediated irradiation protection of the lung and provide a basis for rational design of clinical protocols for radioprotective inhalation gene therapy in patients.